Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution

Yixin Cen,Thomas S Moody,Manfred T Reetz,Mamatjan Arkin,Jiahai Zhou,Qi Wu,Meilan Huang,Warispreet Singh

doi:10.1038/s41467-019-11155-3

Abstract

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105−/His224+ rather than a concerted process.

Highlights

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design
We demonstrate this experimentally, and provide an up to date theoretical analysis why the Ser to Cys exchange causes extreme activity reduction, while additional mutations result in high activity when testing substrates which are essentially not accepted by wild-type (WT)
In order to examine the influence of the nucleophile exchange Ser → Cys on Candida antarctica lipase B (CALB) activity, we selected ester 1 as the model substrate, which is hardly accepted by WT due to steric hindrance of the bulky benzyl group

Summary

Introduction

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-HisAsp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4nitrophenyl benzoate, and tolerating bulky substrates. According to sequence alignment and phylogenetic analysis of proteases, the nucleophile exchanges Ser → Cys and Cys → Ser occurred during the evolution of both serine and cysteine proteases from common ancestors (Supplementary Fig. 1). We demonstrate this experimentally, and provide an up to date theoretical analysis why the Ser to Cys exchange causes extreme activity reduction, while additional mutations result in high activity when testing substrates which are essentially not accepted by wild-type (WT). Structural, kinetic and theoretical investigations point to a distinct catalytic mechanism, different from all serine-lipases known to date

Methods

Results

Conclusion